This case–control study aimed to compare lead (Pb), cadmium (Cd), and arsenic (As) levels in neonates with respiratory distress syndrome (NRDS) with those levels in normal neonates and tested their associations with the severity of NRDS indicated by the levels of serum surfactant protein D (SP-D) and cord blood cardiac troponin I (CTnI), and high-sensitive C-reactive protein (hs-CRP).The study included two groups: G1  and G2 (100 cases with NRDS). Cord blood Pb, erythrocytic Cd (E-Cd), neonatal scalp hair As (N-As), maternal urinary Cd (U-Cd), and arsenic (U-As) were measured by a Thermo Scientific iCAP 6200, while CTnI, hs-CRP, and SP-D by their corresponding ELISA kits.p = 0.019, 0.040, 0.003, 0.010, 0.011, < 0.001, 0.004, < 0.001, respectively). While the birth weight, and APGAR score at 1, 5 and 10 min were significantly lower in G2 than G1 . The levels of the studied heavy metals correlated positively with the levels of SP-D, CTnI, and hs-CRP.The levels of cord blood Pb, E-Cd, N-As, U-Cd, U-As, SP-D, CTnI, and hs-CRP were significantly higher in G2 than G1 (Heavy metals toxicity may be accused to be one of the causes of NRDS especially if other apparent causes are not there. Measuring and follow-up of heavy metal levels should be considered during pregnancy. Neonatal respiratory distress syndrome (NRDS) is a significant cause of newborn mortality that is characterized by lung immaturity and a deficiency in lung surfactant . It affeThe lung surfactant is mostly lipid-dense, consisting mainly of phospholipids, with smaller percentages of neutral lipids and proteins . SurfactExposure to heavy metals is inevitable as they are widely used in industry, households, agriculture, cosmetics, and medical preparations . The lunThe current study aimed to compare the levels of cord blood Pb, cord erythrocytic Cd (E-Cd), and neonatal scalp hair As (N-As) in neonates with NRDS with those levels in normal neonates. Also, maternal urinary Cd (U-Cd) and urinary arsenic levels (U-As) were measured and compared in both groups. In addition, the study tested the associations of these levels with the severity of NRDS indicated by the levels of serum SP-D and cord blood the stress biomarkers; cardiac troponin I (CTnI) and high-sensitive C-reactive protein (hs-CRP).2 (hypercapnia), and acidosis, and the radiological findings [The current work is a case–control study that was conducted between August 2021 and April 2022 in the medical biochemistry department, Assiut University, in cooperation with the Assiut university children’s hospital and with the University Woman hospital. The study included 160 neonates divided into two groups; G1  and G2 . After excluding babies with the exclusion criteria mentioned below, the diagnosis of the NRDS was depending on the clinical manifestations , laboratory analysis (low blood oxygen (hypoxia), elevated arterial COfindings , 13.The neonate was excluded from the study when exhibited congenital anomaly, infections, or inflammation, any other systemic diseases, or signs of hypoxic-ischemic encephalopathy (178 infants were excluded according to these criteria).Complete medical and obstetric histories were taken from the mothers. For neonates, full medical examination, anthropometric measurements, and APGAR scores at 1, 5, and 10 min after birth were done.Five ml of freshly voided midstream urine samples (or by catheter) were collected aseptically. The samples were centrifuged to get rid of sediments then a drop of HCl (conc.) was added to stop any bacterial growth. Then the samples were kept at -70 °C till the measurement the U-Cd and U-As levels by an inductively coupled plasma-optical emission spectrophotometer (Thermo Scientific iCAP 6200) . The alkThe umbilical cord was clamped immediately after delivery, adequately cleaned with an antiseptic agent, and cut. Six ml of cord arterial blood was withdrawn. Two ml of them were left to clot for 20 min, centrifuged, and the serum was separated and kept at -70 °C till the measurement CTnI by the Dimension® (RXL-Max/Xpand) clinical chemistry system with the electrochemiluminescence immunoassay kit (Cat. No: RF421C) and hs-CRP by the ADVIA Chemistry using the kit (Cat.NO:05006455). Both kits were supplied by Siemens Healthcare Diagnostics Products GmbH, Marburg, Hessen, Germany.The remaining 4 ml were divided between two EDTA-containing tubes; Two ml were kept as whole blood and the other 2 ml as RBCs concentrate . The whole blood and RBCs were digested by the method described by Marouf (2011) . Briefly3 (65%) was applied to 0.1 g of hair samples in graduated polypropylene tubes for 24 h. Half ml of H2O2 (30%) was added to the tubes. The final digested samples were diluted to 10 ml by ultrapure deionized water. After cooling to room temperature, the samples were ready for the measurement of N-As by Thermo Scientific iCAP 6200 [Stainless steel scissors were used to cut hair samples from the back of the newborn’s head and collected into a labelled polythene bag, sealed with a zipper. They were cut into small pieces, cleansed, and rinsed five times with deionized water. Washed samples were oven-dried for 24 h at 60 °C before being weighed. For digestion, 2 ml of HNOCAP 6200 , 18.Two ml of venous blood were obtained from each neonate, left to clot for 20 min and centrifuged at 3000 rpm for 15 min. The sera were separated and collected into labeled Eppendorf tubes and frozen at -70 °C. The level of SP-D was measured using human SP-D ELISA kit , after the manufacturer’s instructions.rho) [p-Value ≤ 0.05 was adjudged to be statistically significant [The collected data were examined by SPSS (v.26). After testing the data normality, Mann Whitney U and Chi-square test were used to compare the quantitative and qualitative variables in the two groups . The corrho) . The recrho) . p-Valuenificant .The levels of cord CTnI, hs-CRP, cord blood Pb, E-Cd, N-As, SP-D, U-Cd, and U-As were significantly higher in neonates suffering from NRDS (G2) than the healthy control (G1). While the birth weight, and APGAR score at 1, 5 and 10 min were significantly lower in G2 than G1.p = 0.27).The percentages of preterm infants, exposure to passive smoking, insecticides, pesticides, and other possible sources of heavy metals were higher in G2 than G1. Non-significant differences were found regarding maternal residence and neonatal gender between the two groups (Table p < 0.001) (data not shown in table).A significant lower percentage of mothers who had a history of using antenatal steroids was found in G2 (17%) than those in G1 (70%)  and the N-As levels correlated positively with the maternal U-As  (data not shown in the tables).The cord E-Cd levels correlated positively with the maternal U-Cd levels  (data not shown in the tables).Cord blood Pb levels correlated positively with the levels SP-D, CTnI, and hs-CRP in whole study sample and in G2. Cord E-Cd levels correlated with the levels of CTnI and hs-CRP in whole study sample and only with the levels of CTnI in G2. The N-As and the maternal U-As levels correlated positively with the levels of SP-D and hs-CRP in the whole study sample but only with the levels of SP-D in G2. Maternal U-Cd levels correlated positively with the levels of SP-D in the whole sample. The levels of SP-D were correlated positively with the levels of hs-CRP in the whole sample . Cord E-Cd correlated negatively with the birth weights, and APGAR score at 1 and 5 min . The levels of N-As correlated negatively with the birth weights, and APGAR score at 1 and 5 min . Maternal U-Cd correlated negatively with APGAR score at 10 min . Maternal U-As correlated negatively with birth weights, and APGAR score at 1 and 5 min . SP-D correlated negatively with birth weights and APGAR score at 1 min . The levels of CTnI correlated negatively with birth weights, and APGAR score at 1 and 5 min . The levels of hs-CRP correlated negatively with birth weights, and APGAR score at 1 and 5 min .Also, in the whole study sample, Cord blood Pb levels correlated negatively with the birth weights, and APGAR score at 1 and 5 min , a disrupted production of the fetus's pulmonary surfactant, decreased lung weight and increased postnatal mortality due to respiratory distress were noticed after giving Cd pregnant rats .Acute Pb poisoning impairs surfactant synthesis and disrupts the lung parenchyma and normal respiratory function . The preTurker et al. (2013) reported that lead and cadmium levels in meconium were associated with a high percentage of NRDS and neonatal mortality. These levels were found to be higher in non-surviving neonates than in surviving neonates and the incidence of NRDS and intrauterine growth retardation was significantly high in non-surviving neonates . On the In the current study, the high levels of heavy metals in the maternal and neonatal samples in the NRDS group may be partially attributed to the maternal exposure to passive smoking, insecticides, and pesticides which are known sources for these toxins .Surfactant protein D belongs to the collectin group of defense lectins. Because of its surface tension-lowering properties and vital role in innate immunity, it is important for lung health and optimal respiratory function . In earlThe present study found significant higher levels of blood SP-D in neonates suffering from NRDS than healthy control and these levels correlated with cord blood Pb, N-As and maternal U-As, and maternal U-Cd. Especially in preterm newborns, high serum SP-D is linked to respiratory diseases including RDS . These iIn contrast to our results, Ider et al. (2021) reported lower serum SP-D levels in NRDS than the healthy neonates . Also, HThe stress biomarkers such as CTnI, and hs-CRP have been considered as biomarkers for neonatal stress and indicators of perinatal asphyxia . The sigOn the contrary to the result of the current study, Sorokin et al. (2014) found non-significant association between serum cord blood CRP and development of NRDS, and also reported that the logistic regression analysis revealed that lower risks of RDS were associated with higher umbilical cord serum CRP concentrations in neonates with 32 GA .Regarding ROC analysis, except for the hs-CRP, none of the studied parameters showed a good AUC, sensitivity, or specificity to differentiate neonates with RDS from healthy control. Unfortunately, we found no similar studies in this regard to compare with them.The main limitation of the study is being of a small size.Trials should be taken toward minimizing exposure to heavy metals sources as heavy metals toxicity may be accused to be one of the causes of NRDS especially if other apparent causes are not there. Also, we recommended to monitor cadmium and arsenic levels in risky cases during pregnancy.The main limitation of the current study is the inclusion of both preterm and full-term babies in both the control and the NRDS groups and the dependence on the biochemical data only in the determination of the severity of the NRDS. Future research should consider the preterm infants only in both groups and depend on both clinical and laboratory data for determining the severity of the NRDS.


Caenorhabditis elegans to simulated microgravity and observe the maintained transcriptomic response after returning to ground conditions for four, eight, and twelve days. We show that 75% of the simulated microgravity-induced changes on gene expression persist after returning to ground conditions for four days while most of these changes are reverted after twelve days. Our results from integrative RNA-seq and mass spectrometry analyses suggest that simulated microgravity affects longevity-regulating insulin/IGF-1 and sphingolipid signaling pathways. Finally, we identified 118 genes that are commonly differentially expressed in simulated microgravity- and space-exposed worms. Overall, this work provides insight into the effect of microgravity on biological systems during and after exposure.Given the growing interest in human exploration of space, it is crucial to identify the effects of space conditions on biological processes. Here, we analyze the transcriptomic response of  Significant efforts are being made for the human exploration of space. It is known that space conditions can negatively affect biological processes by causing bone loss, muscle atrophy, and immune system impairment, to name a few examples . HoweMicrogravity is one of the major stress factors causing detrimental health effects on humans (reviewed in ). SimulaC. elegans is an exceptional model organism for space biology studies [C. elegans Experiment in Space (ICE-First), C. elegans RNAi space experiment (CERISE), Shenzhou-8, and Commercial Generic Bioprocessing Apparatus Science Insert-01 (CSI-01)) have provided valuable insights into the biological response of C. elegans to space conditions [ studies  with its studies ). The usnditions ,22,23,24nditions . We prevnditions . Similarnditions . Thus, cC. elegans transcriptome. Through the RNA-sequencing (RNA-seq) and mass spectrometry analyses, we reveal the downregulation of the sphingolipid signaling pathway under simulated microgravity. In addition, we identify a putative microgravity-responsive transcriptomic signature by comparing our results with previous studies.This study investigates the effect of simulated microgravity and the sustained impacts after return to ground conditions on C. elegans were acclimated to CeHR medium for three weeks prior to microgravity experiments. The ground control animals were maintained in CeHR medium in 20 mL scintillation vials as described [0 generation) were cleaned by sucrose floatation, their embryos (F1 generation) were harvested by bleaching and placed in ~40 mL of CeHR culture in sterile VueLife culture bags . 2” and subsequent generations were approximated by using four-day intervals. Prior to RNA extraction, only live worms are retrieved using the sucrose floatation method. We note that “F2” generation and beyond will not maintain synchronicity, and all will contain a mixture of generations, but recovering only the live animals enriches the RNA pool for the expected (majority) generation as each adult will produce ~30 offspring. The reason for the usage of mixed stage worms were the difficulties experienced  during the synchronizing of the animals.Wild-type N2 strain was obtained from the Caenorhabditis Genetics Center (CGC). The worms were grown at 21 °C. Stocks of escribed . ApproxiC. elegans were acclimated to CeHR medium for three weeks prior to microgravity experiments. Approximately 100,000 live worms (P0 generation) were cleaned by sucrose floatation, their embryos (F1 generation) were harvested by bleaching and placed in ~40 mL of CeHR medium in sterile VueLife culture bags . Four culture bags were mounted in a lab-built clinostat inside a laminar flow biosafety hood, and one was kept aside as a ground control. The clinostat rotated the culture bags at 1 rad/s (~10 RPM) for four days, which is the measured time to reach L4/adulthood at 21 °C in CeHR medium [Wild-type N2 strain was obtained from the Caenorhabditis Genetics Center . The worms were grown at 21 °C. Stocks of R medium . Worms fp-values < 0.05, and log2 fold change > 2 unless otherwise stated. The results from the Tuxedo pipeline were used throughout the paper as explained in the results and the results from DESeq2 were reported in the Quality control on the RNA-seq data was done with FASTQC (version 0.11.2) . All thehttps://www.genome.jp/kegg/tool/map_pathway1.html (accessed on 1 January 2019)). Tissue enrichment analysis was performed by using WormBase Gene Set Enrichment Analysis tool [p-value  <  0.05 considered significant and top ten tissues were reported.Gene ontology (GO) enrichment for biological processes and protein domains were assessed with Database for Annotation, Visualization and Integrated Discovery (DAVID) (v6.8) . The GO sis tool , and FDRhttps://genelab.nasa.gov/ (accessed on 1 January 2019)) platform was used to acquire the Gene Expression Omnibus (GEO) accession number for the previous studies on microgravity related gene expressions in C. elegans. We identified four such studies with the following GEO accession numbers: GSE71771 , GSE71770 (four-day CERISE), GSE27338 (eight-day CERISE), and GSE32949 by UKM (PRJNA146465) from the query made on 30 June 2016 with “C. elegans” and “microgravity” terms on GeneLab. We used data from only four-day CERISE as the duration of the study is the same with ours. We analyzed the publicly available microarray datasets with GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/ (accessed on 1 January 2019)) for gene expression under microgravity at the ISS versus 1G control in four day CERISE. The genes with logFC > 2 and Benjamini and Hochberg corrected p-value < 0.05 were considered as differentially expressed.NASA’s GeneLab  [STRING database (version 10.5)  was usedn 3.4.0) . Edge win 3.4.0) .m/z to fragments and retention time. Quantification was performed by ratio of analyte to internal standard response multiplied by ISTD concentration and m/z response correction factor. The results are expressed as ng/rnl. The consistency among the replicates were examined with Pearson correlation. All but one replicates in both the experiments showed high correlation (R2 > 0.9). The ground control replicate which did not show a strong correlation (around 60%) with the other replicates was excluded from the analysis. The data were analyzed with t-test (two-tailed), and p-value < 0.05 was considered significant. Equal variances were confirmed with Levene’s test at α = 0.05 for all the ceramides tested.The worms were chopped manually with razor blades on ice-cold glass. Three replicates from mixed stage worms grown in ground control and simulated microgravity conditions were used simultaneously. Each sample was sent to Avanti Polar Lipids Analytical Services as a frozen extract in glass tubes for liquid chromatography with tandem mass spectrometry (LC/MS/MS) experiment for detection of Ceramide levels. Each sample was extracted by modified Bligh and Dyer extraction . The samC. elegans embryos during clinorotation. The trajectory of a spherical particle in a rotating vessel has been described previously in literature [ω, rad/s), and a radial motion due to centrifugal forces, the velocity of which is proportional to ω2 [We used a laboratory-built clinostat with an integrated microscope to observe the motion of small objects including microspheres, yeast cells, and terature ,11,42,43λ is the viscous drag coefficient, m is the mass of the object, wm is the mass of the displaced water, t is time, g is the acceleration due to gravity, and q is the rotating reference frame defined as:In the above equation, 3 density; Corpuscular Inc., Cold Harbor, NY, USA) suspended in a 3% bovine serum albumin (BSA) solution (n = 8). As a test of the performance of the clinostat we recorded the motion of fluorescent melamine-formaldehyde microspheres .p-value ≤ 0.05 were considered as DEGs. Hundreds of genes demonstrated differential expression during exposure to simulated microgravity and up to eight days after return to ground conditions  in the simulated microgravity-exposed and returned worms against the ground control. The genes with over two-fold log2 expression difference with the FDR-adjusted nditions d. TwelveThe spatial expression of the DEGs is of great importance to identify the potentially affected tissues. Thus, we performed tissue enrichment analysis for the DEGs in simulated microgravity a. The doNext, we conducted pathway enrichment analysis for the DEGs in simulated microgravity and return to ground conditions to identify the altered pathways and whether they remained altered after return to ground conditions b. Our reWe identified the number of simulated microgravity-induced DEGs that preserved their expression patterns after the return to ground control c. The maTo elucidate the genes showing altered expression under simulated microgravity and maintaining these expression patterns after return to ground conditions for short (four days) and long-term (12 days), we categorized the DEGs in Venn diagrams . The genflp-22, flp-8, flp-26, nlp-10, nlp-12, nlp-17, nlp-20, nlp-24, nlp-25, nlp-26, nlp-39, nlp-33, nlp-28, nlp-29, nlp-30, and flp-24) were downregulated during the exposure, and this expression profile was lost after return to ground conditions . Thus, our results did not suggest a significant change in the expression of the genes known to function in muscle morphology.During spaceflight, the neuromuscular system and collagen are negatively affected . In agrepmp-3 presents consistent expression patterns both in our study and previous studies [anr-33, K12G11.14, and ZK822.8 are induced whereas anr-2, anr-9, and Y49A3A.6 are silenced during and 12 days after the exposure.In our previous work, we revealed that the expression of many ncRNA molecules is triggered in response to environmental changes . To iden studies , we constbx-43 is upregulated during the exposure and for at least 12 days after the return to ground. The best human BLASTP-match of tbx-43 in WormBase, TBR, functions in developmental processes and is required for normal brain development . However, many of the upregulated TFs do not have a known function. Hence, our list of simulated microgravity-induced TF gene expressions can be a rich source for the discovery of the microgravity-related transcriptional regulators.We next sought to identify the putative transcriptional regulators  of the simulated microgravity-induced genes and whether these regulators maintain their impact after return to ground conditions. That is, we determined the putative transcription factor (TF) genes  that are2(FPKM) > 1.5 in this analysis. We determined 11 DEGs in the longevity regulating pathway genes most of which are involved in the insulin/insulin-like growth factor signaling pathway (insulin/IGF-1) .Spaceflight affects the mechanisms involved in delayed aging in worms. For instance, Honda et al., found that age-dependent increase in 35-glutamine repeat aggregation is suppressed and seven longevity-controlling genes are differentially expressed in spaceflight ,24. To en/IGF-1) . Interesexpected ,48,49,50expected , and thuhsp-12.6 and extended lifespan (WBRNAi00064044) for sod-3 have been reported. Follow-up experiments are needed to fully understand the role of these genes in the longevity regulation under simulated microgravity.The upregulation of the longevity regulating pathway transcriptional targets generally contributes to increased lifespan. Since many of these targets were downregulated under simulated microgravity, we sought to identify the potential impact of their downregulation by acquiring their RNAi phenotype from WormBase . Lifespap-value < 0.0001). The number of shared genes between the simulated microgravity-induced genes and the genes induced or repressed by DAF-16 are 11 and 35, respectively . Similarly, the number of downregulated DAF-16-repressed genes were insignificant with a total of 57 shared genes   . Togethegba-4) and putative sphingomyelin phosphodiesterase asm-3 are downregulated and these downregulation patterns were sustained for eight days after return to ground conditions . Similarly, sphingoid base (SB) levels decreased 1.9-fold under simulated microgravity, but this decrease was not significant  . Increase in the LacCer and HexCer levels have been determined as biomarkers of aging in humans and murine [C. elegans [To validate that the ceramide and sphingosine levels are indeed decreased in response to the aforementioned gene downregulation, we performed mass spectrometry analysis in ground control and simulated microgravity-exposed worms concurrently b,c. Our = 0.055) b. Intered murine  while La elegans . It is uC. elegans [t-test, p-value < 0.05). It has been found that the inhibition of C24–C26 ceramide-inducing ceramide synthase HYL-1 causes improvements in neuromuscular function and age-dependent hypoxia and stress response [The levels of long acyl-chain ceramides (≥C24) are elevated with advancing age and in age-related diseases such as diabetes and cardiovascular disease while C20 and C26 ceramides are unaffected with aging or developmental stage in  elegans ,57,58. Tresponse ,60. Henc–C22 ceramide functions in resistance to hypoxia [t-test, p-value < 0.05). Collectively, our findings suggest that sphingolipid and insulin/IGF-1 pathways play a role in simulated microgravity response.Along with their roles in aging and longevity, different acyl-chain ceramides have other distinctive functions. For example, C16-ceramide induces germ cell apoptosis  and C20– hypoxia . We deteC. elegans reported in NASA’s GeneLab database (https://genelab.nasa.gov/ (accessed on 1 January 2019)). For this analysis, we used GEO2R to determine the microarray DEGs (|log2 fold change (logFC)| > 2 and FDR < 0.05). We reasoned that the genes that are commonly differentially expressed in all the studies should be the core genes responding to microgravity.To investigate the microgravity-responsive genes, we compared the results from our simulated microgravity experiment to those from four-day CERISE spaceflight experiment on p-value < 0.05)  c. Since  < 0.05) .× 10−16) indicating their collaborative involvement in a biological process  during the mission. In addition, the sphingolipid signaling pathway was differentially expressed in Scott Kelly. The telomere length of Mark Kelly, however, remained relatively stable . TelomerMost of the space flight-triggered physiological changes are reverted after return to Earth ,67. SimiThe limitation of our study is the inclusion of mixed stage worms and the collection of samples from whole animals. This limitation is common among the worm experiments conducted in the ISS ,74,75. CC. elegans and Drosophila melanogaster demonstrated only six common genes from the European Soyuz flights to the ISS [C. elegans strains on gene expression and phenotype [The comparison of spaceflight responsive genes in  the ISS . Similarhenotype ,77 and hGiven the elevated interest in the human exploration of space, it is crucial to determine the detrimental effects of the space conditions on biological systems. We believe that our results will be a valuable reference for the studies on transcriptomic response to microgravity by controlling for the renowned batch effects from space conditions, allowing the worms to acclimate to liquid cultivation before the experiment, presenting the sustained transcriptomic responses after return to ground along with their putative regulators, integrating the results from a previous study conducted at the ISS, and providing the response of different ceramide profiles to simulated microgravity.


Propylthiouracil (PTU) is commonly used to create a model of hypothyroidism, one of the most common diseases worldwide that has a significant social impact. This study aimed to examine the changes in the behavior, cognition, and memory in rats with PTU-induced overt hypothyroidism as well as the effects of tryptophan treatment on this state. For this purpose, we administered 5-OH-tryptophan intraperitoneally or directly into the hippocampus of the hypothyroid animals. Their behavior and cognition were assessed using an open field test, T-maze, and novel object recognition test. There were significant differences in the behavioral patterns of the hypothyroid animals, showing a reduction in locomotor activity, rearing, and memory function compared to the controls. The treatment with 5- hydroxy-tryptophan (5-OH-TRP) alleviated those changes. A staggering amount of research is suggesting that the usual denominators in the pathophysiology of depression and the cognitive decline in hypothyroidism are the hippocampal complex and the distorted serotonin metabolism. In our study, we observed significant beneficial effects on cognitive impairment after 5-OH-TRP administration. Current results are promising and may serve as groundwork for further investigation of functional and structural changes in the hippocampus during a hypothyroid state, and in particular, the effects of serotonin mediation in hypothyroid-associated depressive behavior.Thyroid hormones play an important role in the modeling of neural networks in the brain. Besides its metabolic effects, thyroid dysfunction, and hypothyroidism in particular, is frequently associated with cognitive decline and depressive-like behavior. The current study aimed to examine the changes in behavior, cognition, and memory in rats with propylthiouracil-induced overt hypothyroidism. The behavior and cognition were assessed using the open field test, T-maze, and novel object recognition test. We found significant differences in the behavioral patterns of the hypothyroid animals showing a reduction in locomotor activity, frequency of rearing, and impaired memory function compared to the euthyroid controls. As serotonin is an essential biomarker regulating cognition and mood, we tried to modulate the serotonin mediation in hypothyroid animals through tryptophan administration. Treatment with 5-hydroxy-tryptophan (5-OH-TRP) intraperitoneally for 10 days or directly into the hippocampus as a single injection led to attenuation of the hypothyroidism-induced cognitive and memory decline. A staggering amount of research is suggesting that the common denominators in the pathophysiology of depression and the behavior changes in hypothyroidism are the hippocampal complex and the distorted serotonin metabolism. In our study, it was observed a significant alleviation of cognitive impairment and an improvement of memory performance in hypothyroid rats after 5-OH-TRP administration. Current results are promising and may serve as groundwork for further investigation of functional and structural changes in the hippocampus during a hypothyroid state, and in particular, the effects of serotonin mediation in hypothyroid-associated depressive-like behavior. Hypothyroidism is one of the most common metabolic disorders with varying distribution throughout the world according to local dietary specifics and demographic characteristics. Because of the extensive role of thyroid hormones in the regulation of the metabolism, the symptoms of hypothyroidism are frequently attributed to other diseases ,2. LargeIn our study, we focused on another essential biomarker regulating cognition and mood: serotonin (5-HT), implicated in the pathogenesis of hypothyroidism-associated cognitive impairment ,12,13. IChemically induced hypothyroidism is a well-established model in the literature for studying the depressive-like behavior often found in this state ,20. The All animals were treated in agreement with the general regulations for the treatment of experimental animals, established by the Ethics Committee of the Medical University of Sofia and the Bulgarian Agency for Food Safety , in agreement with EU Directive 2010/63/EU for the protection of animals used for scientific purposes.n-propyl-2-thiouracil (PTU)  for 5 weeks in the ad libitum consumed drinking water [®, St. Louis, MO, USA) in 1% PBS, 5 animals were subjected to stereotactic hippocampal injections of 3 µL solution of 5-OH-TRP (17 mg/mL), and 10 hypothyroid rats were treated with only 1% PBS (either i.p. or by stereotactic injection) for 10 days.Hypothyroidism was induced in 20 male Wistar albino rats with an average starting body weight of about 210 g by administration of 0.01% 6-ng water . After rng water ,24 of 5-https://obsproject.com/). The procedure starts with placing the test animal in the center of the box and tracking the length of the trajectory and the time the animal stays in the inner and outer square over 10 min. The video analysis is performed with specialized ToxTrack software (version 2.93) [The device consists of a box with dimensions of 60 × 60 × 35 cm, divided into two zones—external (60 × 60 cm) and internal (35 × 35 cm) squares and a video camera connected to software recording the movement of the experimental animal  . The innThe device (T-maze) is constructed as an initial alley (15 cm × 35 cm) connected perpendicularly in the middle of an alley measuring 12 cm × 85 cm, forming a T-shape with two arms 12 cm × 35 cm. A plexiglass barrier was placed at both entrances to the shoulders. The test was performed according to Gerlai’s procedure (1998). The rat was first directed to the left shoulder by blocking the entrance to the right one with a plexiglass barrier. When the rat returned to the starting alley after examining the left shoulder, the barrier was removed. The rat was allowed to descend the alley and choose one of the two shoulders. Once the rat selected one arm, the opposite was blocked by the barrier. The free selections were monitored continuously for 10 min which was the total time for research and percentage of alternation .The device is an opaque box (60 × 60 × 35 cm), located in a soundproofed, evenly-lit room. The procedure consists of 3 phases: on the first day—getting used to the new environment for a period of 15 min; on the second day—5 min of training by using two identical objects placed in the box, and the time during which the animal examines them is recorded; phase 3—a 5 min test one hour after the training session. During this phase, one of the already studied objects is replaced with a new one which has a different shape and color. The time for investigation of the familiar and the novel object is taken into account. The objects are selected in order not to resemble food and water and not to have a specific smell. Research behavior is defined as directing the animal’s nose to the object at a distance of fewer than two centimeters, accompanied by a vibration of the whiskers. The ability to distinguish the new from the already known object is presented as the recognition index (RI).After each of the behavioral tests, the apparatus is thoroughly cleaned with 0.1% acetic acid solution to remove olfactory traces. After anesthesia with ketamine and xylazine, an incision was made in the skin of each animal to reveal the skull, and after a careful cleaning of the aponeurosis, the lambda and bregma were detected. Subsequently, and in accordance with stereotactic coordinates ML = 1 mm, AP= −3.4 mm, and DV = 3 mm D.This was followed by the closure of the operative field and follow-up of the animal in a separate clean cage. The animal was subjected to behavioral tests 7 days after the stereotactic injection.p < 0.05 is considered statistically significant. Software used was Sigma Plot 11.0 and GraphPad Prism 9.0.All data from behavioral experiments are presented as means ± SD. Statistical analysis was performed with two-way ANOVA with the following factors: thyroid status (control euthyroid levels and hypothyroidism) and treatment  with Bonferroni post-test. p < 0.001) was observed in the PTU-treated group (0.64 ± 0.36 ng/dl) compared to the control euthyroid < 0.001) in the serum concentration of fT4 levels (p < 0.001) was observed in the PTU-treated group (0.64 ± 0.36 ng/dl) compared to the control euthyroid group (20.40 ± 3.99 ng/dl), confirming the induction of a hypothyroid state.A significant difference (p < 0.05). The i.p. administration of 5-OH-TRP for 10 days and the direct single stereotactic injection in hypothyroid rats normalized their motor activity  = 25.38, p < 0.05; p = 0.084).The experimental data showed that PTU-induced hypothyroidism significantly changed the total horizontal motor activity  = 76.84, p < 0.05), while i.p. treatment and the direct stereotactic injection with 5-OH-TRP significantly and comparably increased the distance traveled in the center  = 53.66; p < 0.05) (p = 0.0002), causing a pronounced anxiolytic effect  = 55.82;  < 0.05)  and incrc effect .p < 0.05), while 5-OH-TRP i.p. treatment for 10 days significantly increased the number of times the hypothyroid animals reared   = 27.52;  < 0.05)  suppressp = 0.0001), whereas TRP administration (either i.p. or stereotactic) relieved amnesia caused by hypothyroidism to levels comparable to euthyroid controls  = 9.397, p = 0.007) (p < 0.05)  = 18.89, = 0.007) . Here, a < 0.05) .p = 0.0001) compared to 78.00 ± 8.367 for euthyroid controls, 73.69 ± 4.159 for euthyroid controls treated i.p. with 5-OH-TRP, 79.09 ± 7.416 for euthyroid controls treated stereotactically with 5-OH-TRP, 68.00 ± 4.472 for hypothyroid animals treated i.p. with 5-OH-TRP, and 68.00 ± 7.583 for hypothyroid animals treated stereotactically with 5-OH-TRP.To study their working memory, the animals were tested for their spontaneous alternation in the T-maze. The difference in the alternation percentage (mean ± SD) of the hypothyroid animals (44.00 ± 5.477) was statistically significant  = 13.66; For our cognitive tests, Wistar albino rats were used to study hypothyroid-associated behavior changes. We have previously published that PTU-induced hypothyroid rats demonstrated a depressive-like behavior tested by the forced swimming test (FST) . AdditioAlthough brain thyroid hormone levels do not correlate with their concentration in the blood, there are reports that the thyroid hormone content in the hippocampus is decreased in Wistar albino and Wistar–Kyoto (WKY) rats receiving PTU .In OFT, we observed that PTU-induced hypothyroidism significantly decreased total horizontal locomotor activity, which confirms several previous reports ,27,28. TAnother important marker of exploration in OFT is rearing, which is one of the most common responses of an animal exposed to an unknown environment. In the current study, we observed significantly reduced rearing in PTU-induced hypothyroid rats, which was reflected by their low exploratory motivation. Sapronov et al. described this phenomenon of decreased rearing in adult rats after thyroidectomy; however, it was found to be not significant . On the A similar pattern was observed in the NOR test. The total time spent exploring the novel object in the NOR test session by the hypothyroid rats was significantly decreased compared to that spent exploring the familiar object, and the total exploration time in hypothyroid rats was delayed in comparison with the euthyroid controls, demonstrating a reduction in short-term memory. On the other hand, there was no significant difference in the time spent exploring the objects by 5-OH-TRP-treated euthyroid rats compared to the untreated control group. These results indicated that the memory formed in the familiarization session was retained for at least 1 h in the group that received 5-OH-TRP. The NOR task evaluates the rodents’ ability to recognize a novel object in the environment. The dorsal hippocampus plays an important role in memory formation, especially when spatial or contextual information is a relevant factor, as in the NOR test ,38.At the same time, untreated euthyroid rats and 5-OH-TRP-treated hypothyroid animals demonstrated similar choice accuracy during the acquisition of T-maze alternation behavior, showing a positive effect of TRP supplementation—either i.p. or intracerebral. Taken together, these data indicate that rats in a hypothyroid state have a deficit in the retention of spatial information for a longer period, which is a process normally dependent on the hippocampus. Data presented in The modulation of tryptophan and 5-HT by thyroid hormones has been well documented in the brain areas of developing hypothyroid rats ,40,41, bOur results confirm previous reports that serotonin mediation can counteract the effects of hippocampal dysfunction caused by hypothyroidism ,44. ThusStudies evaluating the effects of increased hippocampal serotonin metabolism following 5-OH-TRP administration showed enhanced short- and long-term memory, improved learning acquisition, and memory consolidation .A staggering amount of research is suggesting that the usual denominators in the pathophysiology of depression and the cognitive decline in hypothyroidism are the hippocampal complex and the distorted serotonin metabolism. In our study, we explored the behavioral and memory effects of overt hypothyroidism and observed significant beneficial effects on cognitive impairment after 5-OH-TRP administration. Current results are promising and may serve as groundwork for further investigation of functional and structural changes in the hippocampus during a hypothyroid state, and in particular, the effects of serotonin mediation in hypothyroid-associated depressive behavior.


To increase the biodegradation rate and water resistance, the fabricated nanofibrous mats were cross-linked. SEM images showed a uniform and coherent structure of CS-CQD-TiO2-GO nanocomposites and CS-CQD-TiO2-GO electrospun nanofibers mats. FTIR analysis, XRD pattern, SEM mapping, and EDS spectrum demonstrate the accuracy of the synthesis as well as the elemental and chemical structure of the nanofibrous mat. The water contact angle indicated that the nanofibrous mat had a hydrophilic property, which is essential for controlling wound exudates. The tensile strength and elongation tests showed that the nanofibrous mat has suitable mechanical properties for wound dressing, including significant flexibility and strength. Interestingly, antimicrobial testing illustrated that the fabricated nanofibrous mat had antibacterial activity against Gram-negative and Gram-positive bacteria. Appropriate cell viability and cytocompatibility of treated mouse fibroblast NIH3T3 cells with the nanofibrous mat were determined using an MTT assay. The animal study results confirmed the proper potential of the nanofibrous mat in wound dressing applications.Electrospun nanofibrous constructs based on nanoparticles and biopolymers have recently been used in tissue engineering because of their similarity to the extracellular matrix in nature. In this study, electrospun chitosan-carbon quantum dot-titanium dioxide-graphene oxide (CS-CQD-TiO Good proliferation of animal fibroblast cells exposed to compounds containing TiO2 also indicates that it is non-toxic [2 is a photo-catalyst nanoparticle and is activated under the influence of harmful UV irradiation and can only use up to 5% solar energy [2 with carbon-based NPs will improve its separation of electron-hole pairs and move its absorption to the visible portion of the spectrum [2 with metal elements of carbon quantum dots increases the efficiency of nanoparticles [2 and reduce the TiO2 bandgap energy to be activated in the visible light spectrum. The remarkable properties of carbon such as excellent dispersion, biocompatibility, low toxicity, good cell permeability, and easy functionalization with biomolecules have made CQDs applicable in drug delivery systems and wound-dressing [2-CQD compound not only has antibacterial properties but is also a qualified option as medicine for wound healing [2 increases the antibacterial power of TiO2 under solar energy. GO is a two-dimensional carbon-based nanostructure that has received much attention due to its potential of eliciting antibacterial effects, enhancing angiogenesis, improving cell proliferation, and facilitating rapid wound healing [2 is an acceptable option for GO dispersion decoration. Thus, we have used GO in polymeric nanocomposite to accelerate wound healing and enhance cell migration. TiO2-CQD-GO nanocomposites (NC) have the potential of being both antibacterial and non-toxic. However, the use of nanocomposites is much more effective in nanofibers mode since nanofibrous structures exhibit surprising features such as high surface area-to-volume ratio and great porosity [Wound healing issues and subsequent economic and health aspects involve millions of people all around the world . Complexon-toxic . Howeverr energy . Additiospectrum ,8. Combiarticles . CQDs asarticles . This aldressing ,11,12. Tdressing . Interes healing . On the  healing . Further healing . It has  healing . A recen healing . ROS can healing . However healing . TiO2 isporosity . The abiporosity ,23. For porosity . PVA/ZnOporosity .2-CQD-GO NPs to facilitate nanofiber fabrication. Chitosan is a biodegradable and biocompatible polymer that can imitate the structure and function of the natural extracellular matrix. It is one of the many options used to produce biomedical nanofibers [2 NC, which has been used after electrospinning as an effective antibacterial pad to accelerate wound healing.This study used chitosan (CS) polymer as a substrate for TiOnofibers ,27. The nofibers . Since Cnofibers . The reanofibers ,29,30. T3), phosphoric acid (H2PO4), sulfuric acid (H2SO4), phosphate buffer saline (PBS), graphite flakes, KMnO4, fetal bovine serum (FBS), MTT -2,5 diphenyl tetrazolium bromide), H2O2, and DMSO (dimethyl sulfoxide) were brought from Sigma . Diethylenetriamine, anhydrous tetrahydrofuran, thionyl chloride (SOCl2), poly (PVA), and dodecyl amine were bought from Merck, Germany, and the NIH 3T3 fibroblast cells were obtained from ATCC .RPMI-1640, nitric acid  in dark conditions was added to the solution to create cross-links and increase the strength of the composite. Finally, the resulting solution was placed on a stirrer at room temperature for 5 min to obtain a homogeneous solution.For the preparation of CS-GO-CQD-TiO2 NC solution and uniformly stirred for 30 min with a stirrer. With the use of a stirrer, 0.25 g of chitosan was dissolved in 0.6 mL of acetic acid and added to the pre-made nanocomposite solution and, finally, the solution was prepared for electrospinning. The NC solution was fed into a 5 mL syringe with a 22-gauge needle. Then, the syringe was placed in the appropriate position, as well as the attachment of two positive and negative electrodes to the aluminum foil of the collector plate to initiate the flow in the polymer solution. As a result, the electrospinning process began. Then, a syringe with a capacity of 5 mL was filled with the solution and located in the electrospinning setup. A constant flow of solution with a 0.5 mL·h−1 rate, and constant conditions, including 15 kV voltage, 15 cm distance between the collector and tip of the needle, and 28 °C temperature, were applied. The electrospun sample was placed in the open air for 24 h to ensure the evaporation of any solvent.In total, 0.5 g of PVA in 5 mL of DI water was dissolved on a stirrer under vigorous agitation for 1 h, and a homogeneous solution was obtained. The solution was then filled with 350 µL of GO-CQD-TiOThe nanofibrous mats were cross-linked to enhance the mechanical characteristics. Additionally, cross-linking increases nanofiber water resistance ability. Cross-links originate from interactions between the polar hydroxyl groups of PVA, NPs, and chitosan’s amine groups and the aldehyde groups of glutaraldehyde . In thisSynthesized NPs and nanofibers were characterized by zeta potential, Dynamic Light Scattering (DLS), and Scanning Electron Microscope (SEM) techniques. The dry nanofibers were used in order to conduct SEM analysis. Furthermore, the nanocomposites were well dispersed in order to detect the size, zeta potential, and morphology through the mentioned techniques. Other characteristics were defined by Energy Dispersive X-ray Spectroscopy (EDS), SEM mapping, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Diffraction (XRD).2 at room temperature. A maximum of three drops was applied to each sample. Angles were then obtained using Image J: Drop-Analysis software and the mean of three measurements was reported for each sample. These angles indicate the wettability of the solid surface by the liquid [WCA was measured using the sessile-drop method. A specially sized drop  was applied to the sample . The cone liquid ,42.2, the 1 × 1 cm2 fragment of the NC was dried, weighed, and placed in a Petri dish containing 10 mL of PBS with pH = 4.7 and stored at 37 °C for 30 min. Then, it was kept in the oven for 7, 14, 28, 42, 56, 72, and 96 h. Finally, the sample was incubated at room temperature for 3 days to dry completely, and then its dry weight was measured using a digital scale. Degradability was obtained from the following formula [To investigate the biodegradability of CS-GO-CQD-TiO formula :(1)BiodeThe antibacterial activity of the nanocomposites was evaluated by using two indicator bacteria. The characteristics of the bacteria used are listed in 2 was obtained as reported in a previous article [2 at a rate of 100 g·mL−1. The disc was heated for 1 h at 45 °C to dry. E. coli and S. aureus were cultured uniformly with sterile swabs on Neutron Agarculture medium. Then, the CS-GO-CQD-TiO2 disks were placed on the surface of each bacterium at a certain distance from the antibiotic disk. Furthermore, to test the growth inhibition zone made by CS-GO-CQD-TiO2 NC, gentamicin for S. aureus and tetracycline for E. coli were employed as controls. Then, using a ruler to measure the growth inhibition zone’s diameter all around the discs, the data were evaluated using the standard CLSI table.The liquid MIC of CS-GO-CQD-TiO article . The ste2 NC was first extracted. Thus, for each 5 mg, the NC, 1 mL of culture medium was added. After incubation, the samples were added to mouse fibroblast cells at different concentrations of 0.01, 0.06, 0.1 µg·mL−1, and 0.6 at 3 and 5 day intervals. The culture medium was used as a control. In total, 106 NIH3T3 fibroblast mouse cells were cultured in a 96-well plate and then incubated for 24 h at 37 °C to evaluate cytotoxicity by the MTT method. Extracts from each sample were added to the respective wells on a plate, and the NIH3T3 cells were exposed to them for 24 h. Then, 100 mL of colorless PRMI and 10 mL of a 12 mM MTT solution were added to each well after the growth medium had been removed. The supernatant medium was removed after 4 h, and 100 L of DMSO was added to each well. Finally, the amount of OD was calculated at 570 nm. The cytotoxicity test of samples was repeated three times, and the OD was measured in triplicate.CS-GO-CQD-TiOThe mechanical characteristics were evaluated by assessing elongation-at-break and tensile strength with a Texture Analyzer in accordance with the Standard Test Procedure for Tensile Properties (ASTM) standard method . These t2 on cell migration. This test was performed with minor modifications similar to the technique used by Malmir et al. [2 and then incubated for 24 h at 37 °C. There was a 100 µm solution in each well. As a negative control, cells that were treated with the medium were used. At the beginning of the incubations and again after 24 h, the damaged cells were imaged using a Nikon Eclipse Te-2000-U microscope (4X magnification). Using Image J software, the scratch area’s surface A was measured. For each well, the results were calculated using the following formula: 100 × (At24/At0). A Student’s t-test was used in the statistical analysis, which was done using the Sigma Plot software.A scratch wound assay was used to evaluate the effect of CS-GO-CQD-TiOr et al. . First, −1 (body weight) of ketamine and xylazine intraperitoneally while the rats were under anesthesia, the dorsal area of their bodies was shaved from the scapula to the ileum, leaving a shear wound that measured 20 mm × 20 mm. They were then split into 4 groups, non-treated rats, phenytoin-treated rats, PVA-treated rats, and CS-CQD-TiO2-GO electrospun fiber-treated rats. The first day following wound induction, treatment was begun; topically applied polymers were used, and the wounds were treated. On the seventh and fourteenth days, all groups’ wounds were inspected and photographed. Finally, the affected region was cleaned, captured on camera, and estimated with ImageJ software (version 1.44 p). The following formula was used to calculate the wound healing rate [0 and W1 refers to wound area at initial and interval time points, respectively.By injecting 10 mg kging rate :(2)WoundA one-way ANOVA test analyzed the data obtained from the wound area measurement.An identically positioned skin sample was taken from each mouse at the end of the experiment. The tissue samples were embedded in paraffin, fixed in 4% formaldehyde, and cut into slices that were about 4 mm thick. Hematoxylin, eosin (H&E), and TRM were used to stain the sections (Trichrome staining). Their morphological modifications were then assessed.p-value less than 0.05 was considered statistically significant.The experiments in this study were repeated at least three times for each analysis, and the related data were expressed as mean ± standard deviation. ANOVA tests were performed based on groups using GraphPad Prism software. A 2, and CQD-TiO2 nanoparticles in 2 NPs might be a preliminary indication that TiO2 NPs are linked to CQDs. 2, and CQD-TiO2. Particles with a zeta potential greater than 30 mV or less than −30 mV are stable. The reported zeta potential of −60.5 mV for CQD-TiO2 indicates its good stability [Evaluation of DLS test results for pure CQD, TiOtability .2, electrospun PVA, and CS-GO-CQD-TiO2 NC with a magnification of 10,000 times are displayed in 2 NPs were well connected. The roughness seen in 2 containing the PVA. The coherent surface and the obtained nanofibers illustrated the high compatibility of PVA with the NC and a compact structure lacking phase separation [The SEM images of the CS-GO-CQD-TiOparation . The absparation .2, and CS-GO-CQD-TiO2 is illustrated in −1 (C-O), 1200 cm−1 (C-O-C), and 1600 cm−1 (C=O), and are broadband in the range of 3000 to 3700 cm−1. Oxidizing agents have effectively oxidized the graphite as evidenced by the presence of oxygenated functional groups [−1 correspond to the carboxyl group bending vibration and the C=C skeletal vibration, respectively [−1, and the C-O-C bond exhibits vibration at 1163 cm−1. The absence of peaks in 1350–1460 cm−1, which is related to CQD, indicates that CQD-TiO2 NPs are well-synthesized, and these results are similar to the recent papers [2 are well-bonded together. According to relevant studies, chitosan has two firm peaks in 1534 cm−1 and 1640 cm−1, which are related to amide II (N-H bending variations) and amide I (C=O stretching), respectively [−1. The C-H stretch vibrations are thought to be responsible for the peak at about 2900 cm−1 [FTIR analysis of GO, CQD-TiOl groups . Furtherectively . In Figuectively . The C-O900 cm−1 .2-GO-CS and PVA. To facilitate CS-GO-CQD-TiO2 electrospinning, the NC has been combined with PVA [−1 and the C-O bond at 1372 cm−1 belong to PVA. These results are in line with the results of Hassan Adeli et al. [2 mat. It is possible to attribute the O-H peak shift at the composite nanofibrous mat to the creation of hydrogen bonds between the hydroxyl or amino groups in CS and the hydroxyl groups in PVA in GO-CQD-TiO2 and PVA in CS [with PVA . Diana Iwith PVA . Figure i et al. . CharactVA in CS .2, and CQD-TiO2-GO, respectively. According to previously published papers, graphene oxide has a peak in the 2θ ~ 10° region with a base distance of d001 = 6.33 Å, which is used for characterization [2 NPs in 2, and these results are consistent with our previous paper [2, and CQD can be seen in the profile. The weak peak seen at 2θ ~ 10–12° in 2 [2, and GO NPs in the CQD-TiO2-GO NC.rization . As showrization . The XRDus paper . As illu12° in 2 . Therefo2-GO electrospun nanofibrous mats. SEM mapping for PVA was also carried out  measurements. The WCA of CS-CQD-TiO2-GO electrospun nanofibrous mats is depicted in 2-GO nanofibrous mats are hydrophilic. Notably, two essential factors affect the WCA value, namely the physical hydrophobicity of the material itself and the membrane surface roughness [The secretions that are made during an ulcer to the surface of the wound create a favorable environment for the development of bacteria . One of oughness . For exaoughness .The produced nanofibrous mats should have an adequate degradation rate to provide suitable dressing during the wound healing process . Figure 2-GO NC with PVA might offer greater tensile strength and elongation at break than the CS-CQD-TiO2-GO NC  [The nanofibrous mats should be able to provide suitable mechanical characteristics and flexibility in the electrospun state for improved clinical operation in wound dressing application . The resO2-GO NC a. Notabl.15 MPa) .2-GO electrospun nanofibrous on Matson mouse fibroblast NIH3T3 cells. −1 concentrations based on nanocomposites compared to the control after 24 h. The NC solution was toxic at concentrations of 0.6 and 0.1 μg·mL−1, which after one day caused growth inhibition in fibroblast cells. The NC at the concentration of 0.06 μg·mL−1 had almost no toxic effects (cell viability about 90%) on fibroblast NIH3T3 cells. However, after 24 h, the concentration of 0.1 g·mL−1 promoted the development and proliferation of fibroblast cells, proving the safety of the produced NC at this concentration rather than having toxic effects on the cells. The results demonstrated that CS-CQD-TiO2-GO nanofibrous mats are biocompatible and suitable for tissue engineering applications. The results are consistent with the studies of Hassan Adeli et al. on electrospun PVA-CS nanofibers [It was anticipated that the perfect wound dressing must be significantly biocompatible . An MTT nofibers .Staphylococcus aureus bacteria are the main cause of wound infections [Escherichia coli bacteria are the common cause of burn wound infection [2-GO-Cs NC were 0.2 mg·mL−1 for Staphylococcus aureus and 0.1 mg·mL−1 for Escherichia coli. The paper disc was impregnated with the NC at the above concentrations. Gentamicin and tetracycline discs were used as controls. The results in S. aureus was 12 mm, and the halo diameter around the tetracycline antibiotic disk was 21 mm on average. As shown in S. aureus around the sample and gentamicin antibiotic disk was approximately equal. In addition, the mean halo diameter around the NC disk in E. coli, whereas the mean halo diameter around the tetracycline antibiotic disk was 29 mm. The gentamicin antibiotic and the NC disk’s inhibition zones created in E. coli than Gram-positive S. aureus bacteria, presumably because of their different cell wall structures [Previous studies have shown that fections . Moreovenfection . Therefonfection . The MICructures .2-GO influenced cell migration and invasion. For the controlled cells, considerable cell migration was seen after 24 h, as illustrated in 2-GO, injured NIH3T3 fibroblast mouse cell monolayers swiftly healed the gap over the course of 24 h, just like in the control group.A scratch wound assay was carried out to determine whether the treatment of CS-CQD-TiO2-GO treatment, the wound area was significantly reduced in mice. The CS-CQD-TiO2-GO electrospun nanofibrous mats group demonstrated the best wound healing effect on the 7th day with a 52.102% improvement in comparison to other groups. As shown in 2-GO electrospun nanofibrous mats group showed a 93.13% wound healing effect compared with both positive and control groups and phenytoin (p < 0.0001)  . Moteall 14 days .2-GO nanofiber on the skin of injured mice, the wound skin tissue was examined in four groups after 14 days of treatment. The histology of all samples was examined by staining with H&E and TRM dyes. A similar method was performed by Bolle et al. [2-GO nanofiber and the last group treated with the CS-CQD-TiO2-GO nanofiber containing phenytoin (2-GO nanofiber group containing phenytoin were more comprehensive than in the other three groups. This group also had an inflammatory response within 14 days after wound healing. It gradually decreased in this group, and dead cells in the tissue were much less seen than in the other groups, and creatine formation was observed on the fourteenth day.Different epidermis layers can be well identified in the histological images in e et al.  and Murpe et al. . The neghenytoin . Accordi2-GO mats were fabricated for wound healing application. FTIR analysis, XRD pattern, and SEM morphology indicated proper incorporation of NPs into composite and bead-free and uniform structures of the electrospun nanofibrous mats. Furthermore, the EDS spectrum of nanofibers confirmed the presence of related elements to CS, CQD, TiO2, and GO. The size and zeta potential of CQD-TiO2 were 68.7 nm and −60.5 mV, respectively. The mechanical properties were also investigated and the presence of the ceramic phases played a key role in increasing the tensile strength from around 3.8 to 9.0 MPa. Furthermore, the mats exhibited effective activities to inhibit bacterial growth of both Gram-positive and Gram-negative bacteria and showed proper cytocompatibility. The results of in vivo experiments in mice indicated that the CS-CQD-TiO2-GO composite resulted in better reepithelization and wound healing compared to the control group.In this study, novel electrospun nanofibrous CS-CQD-TiO


To this end, we performed an extensive meta-atlas characterization of cellular identities of the human eye, covering a wide range of developmental stages. The resulting map uncovered previously unknown biomarkers of major retinal cell types and those associated with cell-type-specific maturation. Using our retinal-cell-identity map from the fetal and adult tissues, we systematically assessed the fidelity of the retinal organoids in mimicking the human eye, enabling us to comprehensively benchmark the current protocols for retinal organoid generation.Characterizing cell identity in complex tissues such as the human retina is essential for studying its development and disease. While retinal organoids derived from pluripotent stem cells have been widely used to model development and disease of the human retina, there is a lack of studies that have systematically evaluated the molecular and cellular fidelity of the organoids derived from various culture protocols in recapitulating their   •Extensive meta-atlas characterization of cellular identities of the human eye•Uncovered previously unknown biomarkers of major retinal cell types•Discovered genes associated with cell-type-specific maturation of the human eye•Systematically assessed the fidelity of the retinal organoids in mimicking the human eye Human retinal organoids derived from pluripotent stem cells (PSCs) offer unprecedented opportunities to investigate early retinal development and diseases. Systematic assessment of the retinal organoids to recapitulate the human eye is critical for their successful application. Kim et al. performed an extensive meta-atlas characterization of cellular identities of the human eye to generate a retinal cell-identity reference map. They subsequently developed a computational framework and novel metrics for assessing the fidelity of retinal organoids based on this retinal cell-identity reference map. This has led the field to develop advanced 3D in vitro structures that can recapitulate the physiological, morphological, and spatiotemporal patterns of the developing retina  offer unprecedented opportunities to investigate early retinal development and therapeutic applications such as cell transplantation . Early sg retina .The advances in the retinal organoid field have led to the development of state-of-the-art protocols that allow efficient and rapid formation of retinal organoids comprising all the retinal cell types: ganglion, amacrine, bipolar, horizontal, Müller glial, and photoreceptor . These oin vivo counterpart.An increasing number of studies have begun profiling the human organoids derived from these protocols at single-cell resolution to investigate retinal development and disease . These sin vivo counterparts in terms of cell identity, cell-type proportion, and coverage. Finally, we developed a user-friendly application called Eikon (https://shiny.maths.usyd.edu.au/Eikon/) that helps users assess the fidelity of their retinal organoids.Here, we performed an extensive curation of single-cell RNA-sequencing (scRNA-seq) datasets from human retinal tissue and organoids derived from a variety of differentiation protocols, generated a comprehensive map of retinal cellular identities of the mature and fetal eye, and benchmarked the fidelity of the human retinal organoid models in faithfully recapitulating the human eye. The extensive meta-atlas characterization of the retinal cellular identities enabled the discovery of an array of previously unknown marker genes of retinal cell types and those associated with cell-type-specific retinogenesis. Moreover, these cellular identities resolved by age were used to systematically benchmark the current protocols for their capacity to generate cell types that closely emulate their Single-cell transcriptome profiling was applied to resolve the cellular identities in the retinal tissue A. To crein vivo identities.We visualized the integrated datasets by cell type, dataset, and batch within each dataset using uniform manifold approximation projection (UMAP) C and anaTo resolve genes that underlie retinal cellular identity, we computed a cell-type-specific cell-identity score for each gene by dataset and batch using Cepo, a computational method for detecting cell-identity genes . The cluTo systematically quantify the influence of the total number of batches and the batch source on Cepo-derived cell-identity gene statistics, we conducted three assessments. First, we evaluated the stability of the Cepo statistics by randomly subsampling from 50% to 90% of all data batches for inclusion in the generation of the averaged Cepo scores. We then calculated Pearson’s correlation between these scores against those generated from the entire batches. Our findings show that even with subsampling of up to 50% of the batches, the Cepo statistics remain highly reproducible and stable, showing only 0.01 loss in correlation across the cell types A. Next, Finally, we performed principal variance component analysis  on all pTo uncover potential new markers of cell types, we performed a systematic query search on PubMed on the genes highly ranked by Cepo see . We consk-nearest neighbor (kNN) classifier on different numbers of top genes ranked by Cepo and classifying an independent retina scRNA-seq dataset  to mimic the cellular identities of the mature and (2) the fetal retinal tissue; (3) to recapitulate the developmental stage; (4) to generate the cell-type proportions found in the tissue; (5) to generate all the major cell types in the retina; and finally (6) to generate the smallest amount of off-target cells B. The fuUsing this benchmarking framework, we evaluated the capacity of each protocol to generate cell types that closely mimic the human eye. We first evaluated the similarity between the protocols by comparing the cell-type identity scores generated for each dataset and batch. We found that our in-house-generated retinal organoids  and thosTo perform a cross-laboratory and cross-protocol benchmarking of the human retinal organoids, we applied the six fidelity metrics to the organoid datasets. The cell-identity metric was computed as the average of the cell-type-specific scores described in THY1 confirmed the specificity of this marker to RGCs  or negatively  correlate with the time-resolved cell-identity profiles as a proxy for their developmental relevancy A. Our fiThe metric—the proportion of cell types—was computed from the mature and fetal reference, the latter of which is also subdivided into two references denoting early and late fetal maturation. We found that, again, our protocols excelled among the protocols in generating cell-type proportions that represent those found in the mature human eye and not the fetal eye F. IntrigThe final metric, the proportion of off-targets, was computed for all the samples using scClassify, a multi-scale classification algorithm that identifies intermediate and unassigned cells . The avehttps://shiny.maths.usyd.edu.au/Eikon/) where the fidelity and quality of organoids can be explored, assessed, and scored.Overall, our benchmarking study revealed that our in-house protocols  generatein vivo and in vitro systems.A key contribution of this study is the discovery of potential novel markers of the major retinal cell types and of retinogenesis. To elucidate this array of cell-type-specific markers, we integratively analyzed the scRNA-seq datasets of the fetal and the mature retina. This is now possible with the advent of single-cell technologies, which has enabled the extensive exploration of retinogenesis at a single-cell resolution. For example, recent studies have begun elucidating the trajectories of retinal-derived photoreceptor differentiation, highlighting markers that distinguish rod versus cone specification during development . Our stuin vivo human retina. This has revealed that unguided human retinal organoids generated using our in-house differentiation protocol and that of Cowan et al. were top performers , do not fully recapitulate the developmental stages of the human retina D and 6F:cis-regulatory elements of the developing and mature human retina and showed that human retinal organoids are capable of emulating the DNA accessibility of the human retina  and Pengyi Yang (pengyi.yang@sydney.edu.au).Further information and requests for recourses and reagents should be directed to and will be fulfilled by the corresponding authors, Anai Gonzalez-Cordero (This study did not generate new unique reagents.GSE201356. The code generated during this study is available upon reasonable request to one of the corresponding authors. Eikon (https://shiny.maths.usyd.edu.au/Eikon/) is available as an interactive web application to explore the fidelity of retinal organoids.The sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession no. g for 30 min, and the cloudy interphase containing PBMCs was collected. Two million cells were cultured for 6 days in hematopoietic expansion medium StemSpan H3000, with the addition of EPO, IL-3, dexamethasone, ascorbic acid, SCF, and IGF-1. Following expansion, 200,000 cells were nucleofected using an Amaxa 4D nucleofector with Addgene plasmids. The nucleofected cells were plated in a well of a six-well plate coated with Geltrex matrix and transitioned to Essential 8 medium.HPSI0314i-hoik_1 (RRID:CVCL_AE82) was obtained from ECCAC. UCLOOi017-A-1 was derived from healthy donor peripheral blood mononuclear cells (PBMCs) as described previously . PBMCs w2. Human induced PSCs (hiPSCs) were grown and expanded under feeder-free conditions using Essential 8 medium  on six-well plates coated with Geltrex (Invitrogen) at a concentration of 1:100. The medium was replaced daily and cells were passaged at 70% confluency via 5–10 min of 37°C incubation with Versene solution (0.48 mM) (Life Technologies) to detach clumps of cells. Cell clumps were resuspended at a ratio of 1:6–1:12 in E8 with 10 μM ROCK inhibitor  and seeded in fresh Geltrex-coated six-well plates. For differentiation, hiPSCs were grown to 90%–100% confluency.Cells were incubated at 37°C in 5% COtrans-RA to enhance photoreceptor development. At day 90 and until the experimental endpoint, the medium was replaced with ALT90  and supplemented with 0.5 μM RA. The medium was replaced Monday, Wednesday, and Friday and the were cells maintained at 37°C in 5% CO2.Retinal organoids were differentiated as previously described  with somRetinal organoids were washed with PBS, fixed for 40–60 min in 4% paraformaldehyde prior to incubation in 20% sucrose. After 210 days in culture, organoids were embedded in OCT, frozen in liquid nitrogen, and then cryosectioned at 14 μm thickness. Cryosections were blocked in 5% serum in blocking solution  for 2 h. Primary antibody  diluted Conceptualization, H.J.K., A.G-C., and P.Y.; methodology, H.J.K., M.O-W., A.G-C., and P.Y.; investigation, H.J.K., D.K., A.G-C., and P.Y.; data generation, M.O-W., T.H.L., B.Y.L., R.V.J., A.G-C., and P.Y.; writing – original draft, H.J.K., M.O-W., A.G-C., and P.Y.; writing – review & editing, all authors; supervision, R.V.J., A.G-C., and P.Y.; funding acquisition, R.V.J., A.G-C., and P.Y.


While it has long been established that cell membranes are complex assemblies of proteins and bilayer-forming lipids, the inherent mobility and wide-ranging heterogeneity of the lipids have limited our ability to understand cell-membrane structure at a molecular level. Consequently, little is yet known about the protein-lipid and lipid-lipid interplay that exists in situ. The present study exploits the regular architecture of a cholinergic cell membrane to determine how the phospholipid and cholesterol organization is influenced by the protein surfaces and by differences in cholesterol concentration between the two leaflets. Lipids in the leaflet containing the most cholesterol form an ordered sterol-hydrocarbon “skin.” This hitherto unobserved hydrophobic-core structure has far-reaching implications in terms of how cholesterol-rich membranes are constructed and function. Torpedo cholinergic membrane. The lipids exhibit distinct properties in the two leaflets of the bilayer, influenced by the protein surfaces and by differences in cholesterol concentration. In the outer leaflet, the lipids show no consistent motif away from the protein surfaces, in keeping with their assumed fluidity. In the inner leaflet, where the cholesterol concentration is higher, the lipids organize into extensive close-packed linear arrays. These arrays are built from the sterol groups of cholesterol and the initial saturated portions of the phospholipid hydrocarbon chains. Together, they create an ordered ∼7 Å-thick “skin” within the hydrophobic core of the bilayer. The packing of lipids in the arrays appears to bear a close relationship to the linear cholesterol arrays that form crystalline monolayers at the air-water interface.Cell membranes are complex assemblies of proteins and lipids making transient or long-term associations that have yet to be characterized at a molecular level. Here, cryo-electron microscopy is applied to determine how phospholipids and cholesterol arrange between neighboring proteins (nicotinic acetylcholine receptors) of  Cell membranes are fundamental components of all living organisms, and intense efforts have been made to understand how the constituent proteins and lipids build their complex bilayer structures , 2. Whil3Torpedo to determine its three-dimensional protein-lipid structure. This membrane has a relatively simple composition, most densely populated by a single protein (nicotinic acetylcholine receptor), embedded in a cholesterol-rich phospholipid bilayer (Torpedo synapse (The present cryo-electron microscopy (cryo-EM) study exploits the regularity and high protein content of the postsynaptic cell membrane of  bilayer –13. More bilayer A, the pr bilayer , as it d synapse  and at t synapse . Density synapse , 19.In earlier studies, the density maps were obtained from the tubes by helical reconstruction, using local averaging to combine the three-dimensional data from multiple subsets , 20. HerTorpedo ray. Two density maps, 1 and 2, having slightly different surface lattices but the same tube curvature, were obtained by three-dimensional classification . Both maps were at 5.5 Å resolution  and showed lipids with good definition. A structure of the transmembrane domain of the acetylcholine receptor was also obtained by fitting an atomic model of the detergent-solubilized protein . This membrane-intact structure enabled quantitative estimation of the protein-excluding area available to the lipids at different levels across the bilayer .The tubular vesicles analyzed were from a single  protein  to the cSI Appendix, Fig. S3). The transmembrane (TM) α-helices extend slightly beyond the lipid bilayer on the outer (extracellular) side, and the receptor is framed by a ring of submembrane α-helices, MX, which partially penetrate the bilayer on the inner side. These amphipathic helices reduce the area of surface available to the phospholipid headgroups , but alleviate the increase in free volume potentially available to their hydrocarbon chains by providing room for cholesterol within the underlying hydrophobic core  is similar to the bilobed profiles obtained by X-ray scattering from other cholesterol-rich membranes . Again, the headgroups appear rather featureless. However, further into the interior of the bilayer  an anSI AppSI Appendix, Fig. S6A). Second, their organization—different in maps 1 and 2—is evidently determined by the spaces made available by the different lattices, not directly by the protein surfaces. Third, they are visible within the hydrophobic core over a thickness of about 7 Å  and hence span nearly the entire sterol-occupied portion of the bilayer.The sterol-hydrocarbon arrays have several defining properties. First, they are static molecular assemblies, since the same array-motifs occur in maps calculated independently from half-datasets  , confirming that the ordering is not cryo-induced. Most intriguingly, in the context of the cell membrane, the crystal lattice is built from linear arrays of cholesterol having a periodicity (6.0–6.2 Å), which is very similar to that of the observed sterol-hydrocarbon arrays in regions where they pack side by side  , a polym 100.8°) , 27. How 100.8°) A, a feat 100.8°) C, which  100.8°) D with th by side E.Torpedo and other cholinergic membranes is required to enable the classical physiological transitions associated with rapid switching of receptors between closed (or resting), open, and desensitized states .Torpedo membranes , 19Torpeembranes D. It alsembranes .SI Appendix, Fig. S6), their distinct arrangements to fit the differing small volumes available, and their near 6.2 Å periodicity all point to a cholesterol assembly-driven origin.Under saturating conditions, the cholesterol monomers would be expected to form stable associations and could plausibly polymerize, i.e., self-assemble in linear arrays, as in the monolayer films. Hence, while in the outer leaflet, the lipids are presumably in a fluid state like that of a liquid crystalline phase studied in model membrane vesicles ; in the Of wider significance, the sterol-hydrocarbon layer incorporates only the initial saturated portions of the hydrocarbon chains, which are the same for all common phospholipids. The invariably heterogeneous headgroup and tail portions are not involved. The ordered hydrophobic-core structure described here might therefore be present in protein- and cholesterol-rich cell membranes wherever cholesterol in one of the leaflets exceeds saturating amounts. This could be a common occurrence, since cholesterol typically constitutes 30–40 mol % of the total lipids in animal cell membranes , and manT. marmorata electric organ  at 17 °C for 2 wk.Acetylcholine receptor-rich membranes were isolated from fresh ic organ . TubularCholesterol monolayer films were produced at the air-water interface, using 60 μL (4 mm diameter) water-filled Teflon wells. A total of 5 μL of chloroform containing 1 mg/mL high-purity cholesterol (Sigma) was injected into the bottom of the well. The mixture was incubated in a humid atmosphere at 4 °C for 24 h, allowing the chloroform to evaporate and the cholesterol monolayer to develop. A thin hydrophobic carbon film, supported by a perforated EM grid (Quantifoil R 0.6/1) was placed on the water surface and then carefully withdrawn, bringing the cholesterol monolayer with it. The grids were stored in liquid nitrogen before examination by cryo-EM or used directly in room-temperature experiments.V on a FEI Titan Krios electron microscope (Thermo Fisher Scientific), using a Falcon 3 direct-electron detector operating in integrating mode. Underfocus values ranged from 1 to 2.8 μm. The calibrated pixel size on the specimen was 1.34 Å. The total dose was 40 e Å−2, fractionated from 79 frames. Micrograph frame stacks were drift corrected and dose weighted using MotionCor2 (CTFs) were estimated from the aligned, nondose-weighted micrographs using Gctf  was based solely on the  helical family . A value for the regularization parameter T = 10 was applied throughout, except in the second round, where its value was increased to 20 to ensure that details associated with the lipids contributed significantly to the refinement.Analysis of the tubular vesicles . Fitting of the atomic model of the receptor, calculation of projected densities from the cholesterol crystals, and preparation of the structural figures were done using DireX (For ng DireX , UCSF Chng DireX  and PyMOng DireX . Supplementary File